ABSTRACT The prevailing view is that enhancement of dopamine (DA) transmission in the mesocorticolimbic system underlies the rewarding properties of alcohol and nicotine (NIC). The mesolimbic DA system consists of DA neurons in the midbrain ventral tegmental area (VTA) that innervate the nucleus accumbens (NAc). Dopamine neurotransmission is regulated by inhibitory VTA GABA neurons, whose excitability is a net effect of glutamate (GLU) and GABA neurotransmission that are modulated by NIC cholinergic receptors (nAChRs) on afferent terminals. We have shown that these neurons are excited by low-dose ethanol (Steffensen et al., 2009), but inhibited by moderate to high-dose ethanol (Gallegos et al., 1999; Ludlow et al., 2009; Steffensen et al., 2009; Stobbs et al., 2004; Yang et al., 2010), and adapt to chronic ethanol (Gallegos et al., 1999), evincing marked hyperexcitability during withdrawal. Based on our previous studies and data presented here, we propose that VTA GABA neurons are a common substrate for the acute actions of ethanol and NIC. The core thesis underlying this proposal is that 6*-nAChRs on GABA terminals mediate acute ethanol inhibition of VTA GABA neurons and DA release in the NAc. In addition, VTA GABA neuron hyperexcitability during withdrawal from chronic ethanol results from adaptations in presynaptic 6*-nAChRs and postsynaptic GABA(A)R-mediated inhibitory synaptic transmission to these neurons, which contributes to the dysregulation of mesolimbic DA homeostasis that accompanies dependence on ethanol and co-dependence on NIC. We will study of the role of 6*-nAChRs in acute and chronic effects of ethanol on VTA GABA neurons and on DA release. Our proposed studies constitute a focused investigation into the role of 6*-nAChRs in mediating acute ethanol effects on these neurons and their adaptation with alcohol dependence. Our studies will test the following specific hypotheses: 1) Acute ethanol inhibition of VTA GABA neuron activity and phasic DA release results from enhancement of GABA release via 6*-nAChRs on GABA terminals; 2) Lack of 6*-nAChRs results in disrupted ethanol consumption and reward; and 3) Hyperexcitability of VTA GABA neurons during withdrawal from chronic ethanol results from adaptation of 6*-nAChRs and subsequent reduction of DA release at terminals in the NAc. To test these hypotheses, we propose three Specific Aims, which involve electrophysiological, behavioral, neurochemical and molecular experiments with acute and chronic ethanol exposure in GAD GFP knock-in mice, and in wild type (WT) and 6*-nAChR KO mice: 1) Define the role of 6*-nAChRs in acute ethanol actions on VTA neurons and dopamine release in the NAc; 2) Define the role of 6*-nAChRs in mediating ethanol consumption and reward; and 3) Define the role of 6-nAChRs in mediating the hyperexcitability of VTA GABA neurons and lowered dopamine release in the NAc during withdrawal from chronic ethanol. We will show preliminary evidence that 6*-nAChRs mediate ethanol enhancement of NIC currents in recombinant nAChRs expression systems,that ethanol enhancement of GABA inhibition to VTA GABA neurons and ethanol reduction in DA release in the NAc, and compromised ethanol reward in 6*- nAChR KO mice. The proposed studies constitute a thorough and systematic investigation into the role of VTA GABA neurons in mediating the acute effects of ethanol and NIC and the role of 6*-nAChR in modulating GABA neurotransmission to these neurons that critically regulate DA neurotransmission in the mesolimbic system implicated in alcohol reward and dependence. Results from this study could provide a preclinical pharmacologic rationale for considering drugs that act selectively on 6*-nAChR as putative therapeutic agents for the treatment of alcohol dependence and alcohol and NIC co-dependence.